Beta-adrenergic receptor (betaAR) signaling plays an important role in the regulation of cardiac performance. BetaAR downregulation and desensitization resulting in reduced responsiveness to catecholamines are hallmarks of the failing heart. In human heart failure, diminished receptor number (downregulation) and impaired coupling (desensitization) of betaARs result in reduced responsiveness to catecholamines. The process that initiates desensitization of the receptor begins with the phosphorylation of betaARs by beta-adrenergic receptor kinase1 (betaARK1), followed by the binding of beta-arrestin, which together inhibits coupling to G protein and loss of effector (adenylyl cyclase) signaling. The beta-arrestin receptor complex is then targeted to the clathrin-coated pits for endocytosis through a process involving interaction of beta-arrestin with AP-2 and clathrin. Recently we have shown that betaARK1 interacts with the lipid kinase, phosphoinositide 3-kinase (PI3K), to form a cytosolic complex. We have shown that the betaARK/PI3K interaction is critical for the agonist-mediated internalization of betaARs and if the betaARK/PI3K interaction is disrupted in vivo, betaARs do not undergo downregulation and desensitization with chronic catecholamine stimulation. Importantly, PI3K contains both lipid kinase and protein kinase activity. Although we have shown that the lipid products of PI3K are important for receptor internalization, it is not yet known whether the protein kinase activity of PI3K is necessary for betaAR endocytosis and activation of downstream signaling pathways. Since receptor-localized PI3K plays a critical role in betaAR internalization, knowledge of the distinct roles of the protein and lipid kinase activities in the process of betaAR sequestration, resensitization and signaling would contribute to a better understanding of the regulation of betaAR function in the pathogenesis of heart failure. Our Central Hypothesis is that downregulation of cardiac betaARs, in vivo, can be prevented through inhibition of the local generation of D-3 phosphatidylinositols by PI3K within the receptor complex and preserving betaAR function in the failing heart will lead to an amelioration of heart failure in both small and large animal models of cardiac failure. We will test the hypothesis that the protein kinase activity of PI3K is required for betaAR internalization and downregulation, and that betaAR function will be normal in hearts displaying a "physiological" hypertrophic phenotype compared to hearts that show a "pathological" hypertrophic phenotype. Specific Aims (1) To determine the molecular mechanisms of PI3K-mediated betaAR internalization by studying PI3K mutants that lack either protein kinase activity or lipid kinase activity; (2) To determine whether overexpression of the minimal peptide of PI3K (PIK domain peptide, PIKdp), which acts as a dominant negative in vitro, will prevent betaAR downregulation in vivo and rescue mouse models of heart failure: a) chronic pressure overload (transverse aortic constriction, TAC), b) Calsequestrin overexpression X PIKdp, c) muscle LIM protein knock out X PIKdp; (3) To determine whether overexpression of PI3Kdp in vivo by adenoviral-mediated gene transfer to the heart will prevent betaAR downregulation and rescue two large animal models of cardiac failure: a) AdenoPIKdp injected into a rabbit infarct model, b) the pacing pig heart failure model; (4) To determine whether the mechanism that causes transition from hypertrophy to heart failure is due to the type of load on the heart (exercise vs. pressure) or the chronicity of the load. Chronic TAC will be compared to intermittent TAC and two exercise protocols in mice, together with a comprehensive evaluation of the developing cardiac phenotype.